Phytochrome is a photoreceptor, a pigment that organisms use to detect light. The unique photochromic properties of the phytochrome (Phy) photoreceptors allow them to photoconvert between two meta-stable forms, a red light (R) absorbing Pr form, and a far-red light (Fr) absorbing Pfr form. Phytochromes (Phys) act as light-regulated switches in a number of photosensory processes in plants, bacteria, and fungi.
Biochemically, phytochrome is a protein with a bilin chromophore. Sequence alignments and 3-dimensional structural analysis of the chromophore binding domain (CBD) of the bacteriophytochrome (BphP) from the proteobacterium Deinococcus radiodurans (D. radiodurans) demonstrate that many residues close to the phytochrome's biliverdin IXα (BV) chromophore are conserved throughout the Phy superfamily. This conservation suggests that these residues have been retained through evolution because they play important roles in bilin ligation, the formation and stabilization of Pr and Pfr, and transmitting the light signal to the histidine kinase domain of the protein.
Prior mutational studies have identified a number of amino acids that are important for chromophore incorporation and Phy signaling. For example, the histidine directly preceding the cysteine responsible for covalent bilin attachment has been implicated as necessary for bilin ligation and phototransformation in plant phytochromes (Remberg et al., 1999, Eur. J. Biochem. 266: 201-208). A structurally conserved isoleucine (Ile35 in DrBphP, the bacteriophytochrome from Deinococcus radiodurans) near the N-terminus of the phytochromes has been implicated as important for protein solubility (Bhoo et al., 1997, J. Amer. Chem. Soc. 119: 11717-11718). To date, many phytochrome mutations have been generated via random mutagenesis screens. However, it would be advantageous to genetically engineer phytochrome mutations in a more predictable and logical manner. For instance, one or multiple directed amino acid substitutions can be useful to test the effects of size or charge at conserved sites.
Plant phytochromes can exhibit some fluorescence. Wild-type plant phytochromes, and the PAS-GAF-PHY construct from the cyanobacterial phytochrome (Cph) known as Cph1, are shown to be fluorescent with the non-natural linear tetrapyrrole phycoerythrobilin (Murphy and Lagarias, 1997, Current Biology 7: 870-876). For example, U.S. Pat. No. 6,046,014 describes “phytofluors”, which are fluorescent adducts comprising an apoprotein and a bilin. Isolation of fluorescent Cph1 mutants recovered from a PHY domain mutant library was disclosed in Fischer and Lagarias, 2004, Proc. Natl. Acad. Sci. USA 101: 17334-17339.
Various methods and reporter molecules are available for monitoring gene activity and protein distribution within cells. These include the formation of fusion proteins with coding sequences for reporter molecules (markers) such as beta-galactosidase, luciferase, and green fluorescent protein. Particularly useful reporter is the Green Fluorescent Protein (GFP) from the bioluminescent jellyfish Aequorea victoria, which is frequently used as a fluorescent marker, and is described in U.S. Pat. No. 5,491,084. However, the known reporter molecules have a variety of limitations, including short wavelength of the fluorescence emission and small separation between excitation and emission wavelength maxima. The discovery of novel reporter molecules for monitoring gene activity, protein synthesis, and protein distribution within cells, can provide very useful tools for biotechnology applications. The present invention addresses these and related needs.